Production of antibodies and development of enzyme-linked immunosorbent assays for the herbicide butachlor

Abstract

Specific polyclonal antibodies against the herbicide butachlor [2-chloro- N-(2,6-diethylphenyl)- N-(butoxymethyl)acetanilide] were obtained from rabbits immunized with butachlor-3-mercaptopropionic acid (BMPA), coupled to bovine serum albumin (BSA) and soybean trypsin inhibitor. Competitive enzyme-linked immunosorbent assays with photometric detection based on either immobilized antibody (direct assay) or immobilized antigen (indirect assay) were developed and optimized for quantification of butachlor. The best assay sensitivity was observed using antibodies against BMPA–BSA immunogen and immobilized antigen format, where the limit of detection was 0.02 ng ml −1, the standard curve midpoint (IC 50) was 2.4 ng ml −1 and assay duration was 1.5 h. Antibodies were capable of the specific detection of butachlor with cross-reactivities towards structurally related chloroacetanilide herbicides (alachlor, acetochlor, dimethachlor, metolachlor, propachlor) below 0.3%. Direct and indirect assays are compatible with up to 1–2%, v/v of methanol and ethanol in the analyzed probe. Assay application to the analysis of mineral, ground and surface water resulted in butachlor recoveries varying between 70 and 118% between 10 and 100 ng ml −1 herbicide concentrations.